Protein digestion



April 14, 1942. G, B, AYRES ET AL 2,279,908 PROTEIN DIGESTION OriginalFiled June 16, 1959 cc. of 0/ /V 4650/? Wow ATTORNEY.

Patenteci Apr. 14, 1942 v I .rnorcm DIGESTION Gilbert B. Ayres,Stamford, and Joseph George Niedercorn,

Riverside, American Cyanamid Company,

assignors to New York,

Oonm,

N. Y., a corporation of Maine Original application June 16, 1939, SerialNo.

279,470. Divided and this application Novemher 9. 1939, Serial No.303,554

4 Claims.

The invention relates to the controlled digestion of protein materialsand more particularly to the bating of hides and skins by subjectingthem to the action of tryptic enzymes of Aspergillus jumigatus.

It has been discovered that Aspcrgillus fumigatus produces enzymes whichhave suflicient formly good rate of activity in alkalies atleast up tothe point where the alkalinity reaches a pH proteolytic activity underneutral, acid and alkaline conditions to render them commerciallyattractive in the bating of hides and in similar processes involving theselective digestion of proteins.

It is an object of the present invention to provide these enzymes in theform of a dried proteolytic culture which is suitable for use as a bate.It is a further object to provide a method for the bating of hides andskins using these enzymes as bates, preferably in conjunction with adeliming agent such as ammonium sulfate.

The inventionincludes both the production of these enzymes fromAspergillus fumigatus by inoculation of a suitable nutrient medium andcul tivation of the inoculated medium and the provision of the enzymate(enzyme carrier) in dried form, and also the selective digestion ofproteins and the hating of hides and skins by subjecting the materialsto the action of these proteolytic enzymes.

As illustrative of the proteolytic activity in.

alkaline solution of these enzymes produced by Aspergz'llus fumigatusthere is shown in the single figure of the accompanying drawing a curve,the ordinates of which are given in terms of standard alkali and theabscissae in terms of pH values. The curve was constructed from valuesobtained in digesting a casein substrate (sodium caseinate) with aculture of these enzymes at increasing alkalinities of the sodiumcaseinate solution. The undigested casein was precipitated with astandardized solution of sulfuric acid and sodium sulfate and thefiltrate therefrom, containing the digested casein, titrated with 0.1N-alkali. The tests were conducted by a modification of the procedure ofVolhard and Loehlein for th determination of proteolytic activity ofscribed in Praktikum der Physiologischen Chemie, Part 1, pages 258-259,by Peter Rona, 2d ed., Julius Springer, Berlin, 1931. The curve whichwas plotted for the pH range of 7-10, after sloping ofi from the pointof neutrality, changes sign and ascends until a pH of about 9.5 isreached whereupon it again changes sign and slopes downwardly to the pHof 10. By inspection it will be seen from this curve that the enzymes ofthe present invention manifest a unienzymes de- 'layers' on trays.

.sporulation occurs.

value of about 9.5.

The preparation of these enzymes is as follows: A suitable nutrientmedium in the granular or solid discrete particle condition, such asbran,

moistened with an equal weight of water, is in= oculated with a cultureof Aspergillus fumigatus and the inoculated moist bran spread out inthin The inoculated bran is then incubated in an oven maintained at atemperature of about 30 C. andpreferably at not higher than thistemperature, and at a humidity in the oven such that the atmospheretherein is saturated but does not contain sufiicient moisture to causedeposition of the same onto the bran. The inoculated bran is maintainedin the oven until After incubation for the optimum period the branculture may be thoroughly mixed with 0.2% of cresylic acid in solution,if desired, to improve the wetting out of the bran when-used insolution. The culture or enzymate is then dried at a temperature below45 C., e. g. 40 C., and may be used as such for hating, or an ammoniumsalt, e. g. ammonium sulfate, may be incorporated with the moist massand the mixture then dried. The bran used for the culture may or may notbe sterilized before the inoculation and likewise the culture may or maynot be sterilized, although sterilization of the culture or enzymatedoes not appear necessary at the present time.

The enzyme may be liberated from the dried culture by elution withdilute solutions of various salts, such as ammonium chloride, ammoniumsulfate, sodium chloride, sodium sulfate, etc. and the eluted enzymesused in other fields as digestants.

The proteolytic enzymes may be applied to the bating of hides and skinsin the form of an enzymate in any manner now practiced in the art forthe application of other tryptic bates. One method now in use involveswashing the hides from the dehairing step, and adding an ammonium salt,such as ammonium sulfate, to the water containing the hides in order tolower their pH which is generally very high due to the strongly alkalineconditions under which dehairing takes place. Before adding the ammoniumsalt the hides may be treated for removing lime blast by the addition ofa suitable acid, such as hydrochloric acid, to the water bath containingthe hides. After the pH of the bath has been suitably adjusted, theenzyme bate is added thereto in an amount determined by the enzyme unitstrength of the hate, the kind of hide or skin to he hated. the extentof hating desired, the length of the hating time and the temperature ofthe bating bath.

For-the purpose of illustratiim, there is ,described in the followingexample a methodlor hating of hides with the enzymes of Asperoillus.jumigatus. m

,. Example dehairing bath with water for 15 minutes. at. 70 I". Heat thewashed pack in water to 95 F. and add 5 lbs. of hydrochloric acidthereto, 1 the bath showing red to methyl orange. "After three minutesadd limeto the'bathuntil'it 'lsi slightlypink to phenolphthalein." Tothebath] then add 3 lbs. of ammonium sulfate'a'nd about 1%-.(based on theweight of the kips) of en'- zymate prepared as described above. Run the,paddle' forthe' desired length of bating time with a flnalltemperaturetherein of 85F. Aiterthe 'kipsijhave'beenbatedcool them to 70 F; I

;While the application of these enzymeshas been describedwith particularreference to the hating of hides'andskins their utility is not limitedto this field. On the contrary;

ture of 'peptones, chewing gum, glue, foods, drug's tion is intendedas"illustrativeand not as limitingo! the invention; the

scope gotwhich is de- I lined "by [following claims.

This is, a'diyis'ion ol'our cope'ndinglapplication .SerialNoy2'l9A7016,1939."

- What we=claim'is:. r

' Q0 A-processot hating hides-which comprises subiecting them to theaction. or v e p v 1 ent'inj afdried cultureotthe-mold Aspergillus Wash9. 500 lb. pack of limed kips i'romthe enzymes pres- !umigatus. saidenzymes being characterized by 'f-annilo'rmly high rate o proteolyticactivity un- 15 which comprises subjecting them to the action of"enzymes ot nsp'erqill sjumiaatus in a dried cul- .1- .1'e'-'0IIthemold,- said enzymes being characterizedby a-uniformly hig'h 'r'ate ofproteoiytic acthey mayv a be usedfdn many other processes in which a; Itryptic-enzyme of. high activity is desired, such gas, in de'sizing anddegumming textiles, paper sizing, tenderizing of meat, stripping ofgelatin rfroin photographic films and plates, manufac [deg conditi wit na pH range p to iflbbut 9.5.

processj foi under alkaline conditionsiwithin-apfl range up to about 9.5

tivity under alkalineco'nditions within a pH range'up to about0. 5. w

' 3. A process otbatlng hides which comprises subjectin'gfithem to theaction of enzymes of Aspergillusjum iaatus in a dried, culture of theuniformly high-rate ottproteolytic activity under mold, said enzymesbeing characterized by a alkaline conditions'within'a pH range up toabout 9.5; andfin 'the'presencebran ammonium salt.

4g-A'processot' batingjhides' which comprises subjecting them tothe'action oienzymes of Aspergtllus'zjumigctusin a dried culture of themold. said enzymes I being vcharacterized by a uniformly high rate r ofproteolytic activity under v J alkaline conditions-withinapli range upto about 5- ".9.5j.-and jir' 'theipresence .or ammonium sulfate.

Gnnriain AYRES. aosarrr-a nmpnacoan.

